Safe vaccine production, particular for human administration requires the growth of large quantities of virus produced in high yields from a host system, as well as efficient purification and inactivation methods. Serum or serum-derived substances such as albumin, transferrin or insulin and/or proteins from animal or human sources added to a cell culture medium may contain undesirable agents that can contaminate the culture and the biological material produced therefrom. One of the major concerns for the production of biologicals, such as vaccines or recombinant products, is the potential risk of contaminants such as Bovine Spongiform Encephalopathy (BSE). Furthermore, the World Health Organization (WHO) encourages the use of agents having an inactivating effect on the biological activity of DNA. On the basis of a study on the potential risks associated with biologicals produced in animal cells, the WHO Study Group concluded that levels at up to 10 ng nucleic acid per purified dose can be considered acceptable (1998, WHO, Technical Report Series No. 878), but levels of less than 100 pg per dose would be preferred. However, most viruses used for vaccine production have not been purified to preserve their sensitive biological activity which is critical for efficacy of the vaccine, because efficient purification methods seem to influence the immunogenicity and antigenicity of the virus. In addition, purification methods currently available for virus or virus antigen do not always remove the cell culture contaminants efficiently. Thus, for each particular virus, an appropriate method has to be developed and adapted to large scale.
Ross River Virus (RRV) is a mosquito borne Alphavirus which causes a disease in humans known as epidemic polyarthritis (EPA). It is endemic in Australia, with more than 5000 cases presenting each year. Currently, there is no existing vaccine, and mosquito control programs have not had any noticeable effect in reducing the incidence of disease.
An experimental candidate RRV vaccine previously developed by Yu et al. (1994, Vaccine 12: 1118–1124) and Aaskov et al. (1997, Vaccine 15: 1396–1404) has been demonstrated to protect mice from challenge with live virus. This vaccine is obtained from the supernatant of infected VERO cells cultivated on a small scale basis in Roller bottles in serum-containing medium. The virus-containing supernatant is centrifuged to remove cellular debris and the supernatant is incubated for virus inactivation with Binary ethylenimine (BEI) under alkali conditions at pH 8.5–9.0. The inactivated virus suspension is subjected to sucrose gradient centrifugation, the band of virus of the gradient interface recovered is resuspended in saline buffer and used for immunization studies in mice. However, in the preparation solely subjected to sucrose-gradient purification without any other treatment, cellular contaminants, such as cellular DNA or proteins, can be still present in the virus preparation.
The candidate BEI-inactivated Ross River Vaccine described, supra, is tested at different concentrations of an adjuvant and without adjuvant in mice. Greater protection is found with the vaccine at higher dose without adjuvant than those with an adjuvant. Even at lower dose the non-adjuvanted vaccine induced significantly higher antibody titers compared to the adjuvanted. Comparative studies on neutralizing antibody titers after a second injection of mice with low antigen concentrations of 0.2 or 2.0 μg of vaccine with and without adjuvant caused researchers to conclude that the adjuvant may suppress at low concentrations the protective component of a secondary immune response to the BEI-inactivated vaccine.
Because of the need for a safe and effective Ross River Virus Vaccine for human administration, large-scale production of virus and efficient purification and inactivation methods are required. The present invention is directed to the large-scale production of Ross River Virus suitable for preparation of a vaccine for administration to humans.